Withania somnifera root extract inhibits MGO-induced skin fibroblast cells dysfunction via ECM-integrin interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (L.) Dunal, known as Ashwagandha, has long been used in traditional medicine in Ayurveda, India, a representative adaptogen. The main active constituents of W. somnifera are withanolides, and the root is often used as a medicine with a wide range of pharmacological activities, which can be used to treat insomnia, neurasthenia, diabetes mellitus and skin cancer. AIM OF THE STUDY: Whole-component qualitative and quantitative analyses were performed on W. somnifera. We explored the ameliorative effect of the adaptogen representative plant W. somnifera on the senescence events of MGO-injured fibroblasts and its action mechanism and verified the hypotheses that WS can inhibit the accumulation of AGEs and regulate the dynamic balance among the components of the ECM by modulating the expression of integrin ß1 receptor; as a result, WS maintains cellular behavioural and biological functions in a normal range and retards the aging of skin from the cellular level. MATERIALS AND METHODS: In this study, the components of WS were first qualitatively and quantitatively analysed by HPLC fingerprinting and LC-MS detection. Second, a model of MGO-induced injury of CML-overexpressing fibroblasts was established. ELISA was used to detect CML expression and the synthesis of key extracellular matrix ECM protein components COL1, FN1, LM5 and TNC synthesis; CCK-8 was used to detect cell viability; EDU was used to detect cell proliferation capacity; fluorescence was used to detect cell adhesion capacity; and migration assay were used to detect cell migration capacity; qRT-PCR was used to detect the regulatory pathway TGF-ß1 and MMP-2, MMP-9 in ECMs; immunofluorescence was used to detect the expression of ITGB1; and WB was used to detect the expression of COL1, FN1, LM5, Tnc, TGF-ß1, MMP-2, MMP-9 and ITGB1. RESULTS: In total, 27 active ingredients were analysed from WS, which mainly consisted of withanolide components, such as withaferin A and withanolide A. Based on the model of MGO-induced fibroblast senescence injury, WS significantly inhibited CML synthesis. By up-regulating the expression of integrin ß1, it upregulated the expression of the TGF-ß1 gene, which is closely related to the generation of ECMs, downregulated the expression of the MMP-2 and MMP-9 genes, which are closely related to the degradation of ECMs, maintained the dynamic balance of the four types of ECMs, and improved cell viability as well as proliferation, migration and adhesion abilities. CONCLUSIONS: WS can prevent cellular behavioural dysfunction and delay skin ageing by reducing the accumulation of CML, upregulating the expression of the ITGB1 receptor, maintaining the normal function of ECM-integrin receptor interaction and preventing an imbalance between the production and degradation of protein components of ECMs. The findings reported in this study suggest that WS as a CML inhibitor can modulate ECM-integrin homeostasis and has great potential in the field of aging retardation.

Extracellular Vesicle-Derived circITGB1 Regulates Dendritic Cell Maturation and Cardiac Inflammation via miR-342-3p/NFAM1.

Acute myocardial infarction (AMI) is a complication of atherosclerosis-related cardiovascular illness that is caused by prolonged ischemia. Circular RNAs (circRNAs) are concentrated in extracellular vesicles (EVs) and have been linked to cardiovascular disease. However, additional research is needed into the expression and function of circRNAs in AMI. In this study, circITGB1 (has_circRNA_0018146), derived from exon 1 of the ITGB1 gene localized on chromosome 10, was shown to be considerably increased in plasma from patients with AMI compared to healthy controls, as demonstrated by the comparison of EV-circRNA expression patterns. Using a luciferase screening assay and a biotin-labeled circITGB1 probe to identify microRNA(s) complementary to circITGB1 sequences, we discovered that circITGB1 competitively binds to miR-342-3p and inhibits its expression, which in turn increase the expression of NFAT activating molecule 1 (NFAM1). Based on western blotting and immunological studies, circITGB1 controls dendritic cell maturation by targeting miR-342-3p and NFAM1. circITGB1 also exacerbated cardiac damage and regulated miR-342-3p and NFAM1 expression in a mouse AMI model. This implies that EV-circITGB1 is involved in dendritic cell maturation and cardiac damage via miR-342-3p/NFAM1, and that is linked to AMI-associated pathogenic processes.

Extracellular matrix and Hippo signaling as therapeutic targets of antifibrotic compounds for uterine fibroids.

BACKGROUND: Uterine fibroids are highly prevalent, collagen-rich, mechanically stiff, fibrotic tumors for which new therapeutic options are needed. Increased extracellular matrix (ECM) stiffness activates mechanical signaling and Hippo/YAP promoting fibroid growth, but no prior studies have tested either as a therapeutic target. We tested the hypothesis that injection of a purified form of collagenase Clostridium histolyticum (CCH) that selectively digests type I and type III collagens would alter ECM stiffness, Hippo signaling, and selectively reduce fibroid cell growth. We also used two FDA-approved drugs, verteporfin and nintedanib, to elucidate the role of Hippo/YAP signaling in uterine fibroid and myometrial cells. METHODS: The clinical trial was registered (NCT02889848). Stiffness of samples was measured by rheometry. Protein expression in surgical samples was analyzed via immunofluorescence. Protein and gene expression in uterine fibroid or myometrial cell lines were measured by real time PCR and western blot, and immunofluorescence. RESULTS: Injection of CCH at high doses (0.1-0.2 mg/cm3 ) into fibroids resulted in a 46% reduction in stiffness in injected fibroids compared to controls after 60 days. Levels of the cell proliferation marker proliferative cell nuclear antigen (PCNA) were decreased in fibroids 60 days after injection at high doses of CCH. Key Hippo signaling factors, specifically the transcriptionally inactive phosphorylated YAP (p-YAP), was increased at high CCH doses, supporting the role of YAP in fibroid growth. Furthermore, inhibition of YAP via verteporfin (YAP inhibitor) decreased cell proliferation, gene and protein expression of key factors promoting fibrosis and mechanotransduction in fibroid cells. Additionally, the anti-fibrotic drug, nintedanib, inhibited YAP and showed anti-fibrotic effects. CONCLUSIONS: This is the first report that in vivo injection of collagenase into uterine fibroids led to a reduction in Hippo/YAP signaling and crucial genes and pathways involved in fibroid growth. These results indicate that targeting ECM stiffness and Hippo signaling might be an effective strategy for uterine fibroids.

Smilax glabra Roxb. Inhibits Collagen Induced Adhesion and Migration of PC3 and LNCaP Prostate Cancer Cells through the Inhibition of Beta 1 Integrin Expression.

Smilax glabra Roxb. (SGR) has been used as a traditional medicine for brucellosis and syphilis. In this study, we investigated whether nontoxicological levels of water extract of SGR (WESGR) are effective for suppressing steps in the progression of prostate cancer, such as collagen-mediated migration and adhesion and identified the target molecule responsible for such effects. We found that nontoxicological levels of WESGR did not attenuate PC3 and LNCaP cell adhesion to serum but did significantly do so with collagen. In addition, using the Boyden chamber assay, we found that nontoxicological levels of WESGR did not inhibit the migration of PC3 and LNCaP cells to a serum-coated area but did significantly attenuate migration to a collagen-coated area. Interestingly, the expression of α2ß1 integrin, a known receptor of collagen, was not affected by ectopic administration of WESGR. However, WESGR significantly attenuated the expression of ß1 integrin, but not α2 integrin when PC3 and LNCaP cells were placed on a collagen-coated plate, resulting in attenuation of focal adherent kinase phosphorylation. Finally, 5-O-caffeoylquinic acid was determined as a functional single component which is responsible for antiprostate cancer effects of WESGR. Taken together, our results suggest a novel molecular mechanism for WESGR-mediated antiprostate cancer effects at particular steps such as with migration and adhesion to collagen, and it could provide the possibility of therapeutic use of WESGR against prostate cancer progression.

Garlicin Post-Conditioning Suppresses Adhesion Molecules in a Porcine Model of Myocardial Ischemia-Reperfusion Injury.

OBJECTIVES: To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin ß1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). METHODS: Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion. RESULTS: Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively). CONCLUSIONS: Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.

Irigenin, a novel lead from Western Himalayan chemiome inhibits Fibronectin-Extra Domain A induced metastasis in Lung cancer cells.

Several lines of evidence indicate that Fibronectin Extra Domain A (EDA) promotes metastatic capacity of tumor cells by engaging cell surface α9ß1 integrins. This interaction mediated by the C-C loop of EDA activates pro-oncogenic signaling pathways leading to epithelial to mesenchymal transition (EMT) of tumor cells, thus signifying its importance in control of metastatic progression. In this context the present study was designed to explore the active compounds from selected ethno-medicinal plants of western Himalayan region for targeting EDA of Fibronectin in lung carcinoma cells. Structure based informatics for drug designing and screening was employed to generate a lead compound(s) feed that were conformationally and energetically viable. Out of 120 compounds selected, Irigenin showed best binding-affinity with C-C loop of EDA. Irigenin specifically targeted α9ß1 and α4ß1 integrin binding sites on EDA comprising LEU46, PHE47, PRO48, GLU58, LEU59 and GLN60 in its C-C loop as evaluated by energy decomposition per residue of Irigenin-EDA complex. In-vitro cell motility assays complemented with EDA knock-in and knockdown assays distinctively demonstrated that Irigenin prevents metastatic capacity of lung cancer cells by selectively blocking EDA. The results presented thus project Irigenin as a lead compound to overcome Fibronectin EDA induced metastatic progression in lung carcinoma cells.

Beneficial Effects of the Genus Aloe on Wound Healing, Cell Proliferation, and Differentiation of Epidermal Keratinocytes.

Aloe has been used as a folk medicine because it has several important therapeutic properties. These include wound and burn healing, and Aloe is now used in a variety of commercially available topical medications for wound healing and skin care. However, its effects on epidermal keratinocytes remain largely unclear. Our data indicated that both Aloe vera gel (AVG) and Cape aloe extract (CAE) significantly improved wound healing in human primary epidermal keratinocytes (HPEKs) and a human skin equivalent model. In addition, flow cytometry analysis revealed that cell surface expressions of ß1-, α6-, ß4-integrin, and E-cadherin increased in HPEKs treated with AVG and CAE. These increases may contribute to cell migration and wound healing. Treatment with Aloe also resulted in significant changes in cell-cycle progression and in increases in cell number. Aloe increased gene expression of differentiation markers in HPEKs, suggesting roles for AVG and CAE in the improvement of keratinocyte function. Furthermore, human skin epidermal equivalents developed from HPEKs with medium containing Aloe were thicker than control equivalents, indicating the effectiveness of Aloe on enhancing epidermal development. Based on these results, both AVG and CAE have benefits in wound healing and in treatment of rough skin.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. METHODS: AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. RESULTS: We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin ß1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. CONCLUSIONS: We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

Effects of acupuncture at Baihui (GV 20) and Zusanli (ST 36) on peripheral serum expression of MicroRNA 124, laminin and integrin ß1 in rats with cerebral ischemia reperfusion injury.

OBJECTIVE: To explore the effects of acupuncture at Baihui (GV 20) and Zusanli (ST 36) on the peripheral serum expression of microRNA 124 (miRNA 124), laminin and integrin ß1 in rats with cerebral ischemia reperfusion injury (CIRI). METHODS: Seventy-two healthy male Sprague-Dawley rats were randomized into a model group, an acupuncture group, and a sham-operated group using a random digits table, with 24 rats per group. Each group was further randomly divided into 1-, 3-, 5-, and 7-day subgroups based on the reperfusion time according to a random digits table, with 6 rats in each subgroup. In the model and acupuncture groups, CIRI was induced using the thread occlusion method. Electroacupuncture stimulation was applied daily to GV 20 and left ST 36 for 20 min at the indicated time points after successful operations. Serum was sampled for detecting laminin and integrin ß1 protein via enzyme-linked immunosorbent assay, and serum miRNA 124 was examined using quantitative polymerase chain reaction. RESULTS: The serum level of miRNA 124 in the cerebral ischemia rats increased significantly, and the peak expression of miRNA 124 in both the model and acupuncture groups occurred at 3 days. The expression of miRNA 124 in the acupuncture group was higher than in the model group at the same time point (5.96±0.01 vs. 3.11±0.04, P <0.05). Laminin expression in serum from the cerebral ischemia group was higher than that in the sham-operated group. Compared with the model group, the level of laminin in the serum of the acupuncture group was significantly lower at each time point, especially at the 3-day, and 7-day time points (589.12±3.57 vs. 793.05±5.28, and 600.53±3.05 vs. 899.06±5.74, P <0.05). The level of integrin ß1 in the serum from the acupuncture group was lower than that in the model group particularly at the 3-day and 7-day time points (208.66±0.95 vs. 280.83±1.77, and 212.36±0.95 vs. 316.77±2.42, P <0.05). Additionally, the model group and the acupuncture group showed dual peaks of integrin ß1 and laminin expression at 3-day and 7-day. CONCLUSIONS: Acupuncture at GV 20 and ST 36 in rats alleviated CIRI and was associated with upregulated expression of miRNA 124 and with downregulated expression of integrin ß1 and laminin in peripheral serum. These changes may represent one of the mechanisms underlying acupuncture's attenuation of CIRI.

[Effects of acupotomy intervention on regional pathological changes and expression of carti- lage-mechanics related proteins in rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effect of acupotomy (needle-knife) therapy on local pathological changes and cartilage-mechanics related protein expression in rabbits with knee osteoarthritis (KOA) so as to study its mechanisms underlying improving KOA. METHODS: Forty New Zealand rabbits were randomly divided into normal control group, model group, acupotomy group, and electroacupuncture (EA) group (n = 10 in each group). The KOA model was established by immobilization of the left knee-joint (modified Videman method) for 6 weeks. After modeling, acupotomy relaxing was applied to the lateral collateral ligament and patellar ligament of the left knee-joint, once a week for 3 times, and EA (2 Hz/100 Hz, 3 mA) was applied to the left "Yanglingquan" (GB 34), "Yinlingquan" (SP 9), "Neixiyan" (EX-LE 4) and "Waixiyan" (ST 35) for 20 min, 3 times a week for three weeks. The expression levels of Integrin ß1, type II collagen (Col-II), matrix metalloproteinase (MMP)-3 and Aggrecan proteins of the cartilage tissue of the left femoral medial and external condyles were observed by Western blot. Pathological changes of the knee-joint by X-ray scanning and those of the femoral condyle tissue were evaluated by Mankin's scores under light microscope after H. E. staining. RESULTS: X-ray showed successful modeling, and pathological changes of the articular cartilage belonged to the early and moderate lesion of knee osteoarthritis. The Mankin's score was significantly higher in the model group than in the control group (P < 0.01) , and after the treatment, the Mankin's scores were significantly decreased in the acupotomy. group (P < 0.01), rather than in the EA group (P > 0.05). The results of Western blot showed that after modeling, the expression levels of Integrin ß 1, Col-II and Aggrecan proteins of the femoral articular cartilage were considerably decreased (P < 0.01), while that of MMP-3 protein was significantly increased (P < 0.01). Compared with the model group, the decreased expression levels of Integrin ß 1, Col-II and Aggrecan proteins in the acupotomy group and Integrin ß 1 protein in the EA group were notably up-regulated (P < 0.01 , P < 0.05), and MMP-3 expression in the acupotomy group was significantly down-regulated (P < 0.01). No significant changes were found in the EA group in the expression levels of Col-II , Aggrecan and MMP-3 proteins compared with the model group (P > 0.05). CONCLUSION: Acupotomy intervention can relieve KOA-induced injury of the knee-joint in KOA rats, which is associated with its actions in raising expression levels of Integrin ß 1, Col-II and Aggrecan proteins and in lowering the expression of MMP-3 proteins in the articular cartilage, probably by adjusting the mechanics-related signal pathway of the articular chondrocytes.

Effects of Ganfukang on expression of connective tissue growth factor and focal adhesion kinase/protein kinase B signal pathway in hepatic fibrosis rats.

OBJECTIVE: To investigate the effect of Ganfukang (GFK) on connective tissue growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or Akt) signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK. METHODS: Fifty SD rats were randomly divided into five groups as follows: the control group, the model group (repeated subcutaneous injection of CCl4), and the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric administration). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α5, integrin ß1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats. RESULTS: GFK attenuated the up-regulation of CTGF, integrin α5, and integrin ß1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression of cyclinD1, collagen I, and collagen III was decreased by GFK significantly (P<0.01). CONCLUSIONS: CTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.

Effect of enoxaparin and onion extract on human skin fibroblast cell line - therapeutic implications for the treatment of keloids.

CONTEXT: Keloids and hypertrophic scars are hyperproliferative skin disorders resulting in abnormal wound healing. In the prevention and treatment of keloids and hypertrophic scars, ointments containing heparin and onion extract are very popular. Their therapeutic effects, however, are still controversial and the mechanism of action is not fully understood. OBJECTIVE: The aim of this study was to assess the effect of enoxaparin and dry onion extract on proliferation, apoptosis and ß1 integrin expression in human fibroblasts. MATERIALS AND METHODS: Fibroblast human cell lines (46 BR.1 N) were treated for 48 h with various concentrations of enoxaparin sodium (20, 100, 500 µg/mL) and/or onion [Allium cepa L. (Alliaceae)] extract (50, 250, 1000 µg/mL). The cell proliferation was evaluated by [(3)H]-thymidine incorporation assay. Furthermore, the expression of ß1 integrin and apoptosis was determined by flow cytometry. RESULTS AND DISCUSSION: The results demonstrate that enoxaparin and onion extract inhibited the proliferation of human fibroblasts. Almost complete inhibition of cell proliferation was achieved by enoxaparin in 500 µg/mL concentration (91.5% reduction). The onion extract at a concentration of 250 µg/mL also strongly inhibited the proliferation of cells (50.8% reduction). Depending on concentration, enoxaparin and onion extract induced apoptosis (500 and 1000 µg/mL, respectively) and, depending on concentration, downregulated the expression of ß1 integrin on human fibroblasts. CONCLUSION: This work points at possible mechanism of action of enoxaparin and onion extract, when administered in the treatment of patients with keloids and hypertrophic scars.

Electroacupuncture inhibits annulus fibrosis cell apoptosis in vivo via TNF-α-TNFR1-caspase-8 and integrin ß1/Akt signaling pathways.

OBJECTIVE: To examine whether electroacupuncture (EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis (AF) via tumor necrosis factor-α (TNF-α)-tumor necrosis factor receptor 1 (TNFR1)-caspase-8 and integrin ß1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces. METHODS: Thirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui (GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8. Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The mRNA and protein expression levels of integrin ß1 and Akt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively. RESULTS: Treatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin ß1 and Akt mRNA and protein levels compared with controls. CONCLUSION: Treatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin ß1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.

Decoctions from Citrus reticulata Blanco seeds protect the uroepithelium against Escherichia coli invasion.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional usage suggests Citrus reticulata Blanco seeds have beneficial effects against infection. The purpose of this study was to investigate the effect of Citrus reticulata on the uroepithelium and to determine the mechanisms responsible for protection against urinary tract infection (UTI). MATERIALS AND METHODS: Human bladder cell lines T24 and 5637 were employed in a cell culture infection model to determine the effects of Citrus reticulata treatment on Escherichia coli adherence and invasion of the uroepithelium. ß1 integrin and caveolin-1 mRNA expression was assessed using RT real-time PCR. ß1 integrin protein expression was confirmed by Western Blot. The effect of Citrus reticulata on bacteria was investigated using antibacterial sensitivity, yeast agglutination and biofilm assays. RESULTS: Citrus reticulata treatment decreased ß1 integrin expression and reduced bacterial invasion while adhesion of uroepithelial cells was not affected. Caveolin-1 expression was not influenced either and Citrus reticulata did neither exhibit any direct antimicrobial effect nor interfered with type 1 fimbriae binding. CONCLUSIONS: Our results show that Citrus reticulata has a protective effect on the uroepithelium as seen by reduced bacterial invasion of uroepithelial cells. These properties suggest that seeds from Citrus reticulata may have therapeutic potential in preventing UTI.

Enhanced osteogenic fate and function of MC3T3-E1 cells on nanoengineered polystyrene surfaces with nanopillar and nanopore arrays.

During in vitro culture, cell fate and function, including cell adhesion, morphology, proliferation and differentiation, are affected by surface characteristics, such as geometry, wettability, hardness, chemistry and charge. This study replicated two different types of nanoengineered polystyrene surfaces (NPS) containing nanopillar (NPS-Pi) or nanopore (NPS-Po) arrays by hot embossing and investigated their topographical effects on cell behavior using osteoblast-like MC3T3-E1 cells. To mass-replicate NPS, rigid metal nano-stamps were manufactured by nickel electroforming onto two different nano-templates: (1) a nanopore-arrayed anodic aluminum oxide nano-template using two-step electrochemical oxidation and (2) a nanopillar-arrayed polymer using hot embossing process. The physical and mechanical properties of the NPS, including geometry, wettability, hardness and elastic modulus, were evaluated with the help of field emission-scanning electron microscopy, a contact angle meter, and a nanoindenter. The nanotopography maintained the bulk property, while drastically changing the surface properties. In vitro the NPS had significant effects on MC3T3-E1 cell morphology, attachment, proliferation and osteogenic differentiation compared to a flat substrate due to the altered physical and mechanical surface properties of the nanoengineered surface. Interestingly, the NPS-Po was more effective at enhancing cell proliferation and osteogenesis differentiation. One potential explanation for these results may be that the subcellular binding sites induced by the nanostructures changed the cell morphology and promoted contractile cytoskeletons, thereby enhancing osteogenic differentiation. This, which allows for the cost-effective replication of NPS and the control of cell behavior, has various applications with respect to biomedical and cell surface interaction studies, in addition to enhanced osteogenic cell fate and function.

ß1 integrin-extracellular matrix interactions are essential for maintaining exocrine pancreas architecture and function.

Integrin receptors are responsible for integrating extracellular matrix signals inside the cell. The most prominent integrin receptor, ß1 integrin, has a role in cell function, survival and differentiation. Recently, we demonstrated a profound in vivo role of ß1 integrin expression in the pancreas on glucose homeostasis and islet function. Here, we extend these results by examining the role of ß1 integrin in exocrine pancreatic structure and function. Adult C57Bl/6 mice hemizygous for a collagen type Iα2 (Col1a2) promoter-controlled tamoxifen-inducible Cre recombinase gene and homozygous for loxP-ß1 integrin were injected with tamoxifen or corn oil to generate mice deleted or not for ß1 integrin. Pancreata derived from these male mice were analyzed by quantitative reverse transcriptase-polymerase chain reaction, western blot and immunofluorescence. Our results showed that ß1 integrin-deficient mice displayed a significant decrease in pancreas weight with a significant reduction of amylase, regenerating islet-derived protein II and carboxypeptidase-A expression (P<0.05-0.01). Compared with control pancreata, ß1 integrin-deficient pancreata showed reduced mRNA expression of extracellular matrix (collagen type Iα2, fibronectin and laminin) genes (P<0.05), detached acini clusters and lost focal adhesion structure. Moreover, ß1 integrin-deficient pancreatic acinar cells displayed decreased proliferation (P<0.05) and increased apoptosis (P<0.001). Apoptosis was reduced to that of controls when isolated exocrine clusters were cultured in media supplemented with extracellular matrix proteins. Taken together, these results implicate ß1 integrin as an essential component for maintaining exocrine pancreatic structure and function.

Ginsenoside Rh1 inhibits the invasion and migration of THP-1 acute monocytic leukemia cells via inactivation of the MAPK signaling pathway.

Ginsenoside Rh1 has been reported to possess antiallergic and anti-inflammatory activities, but its effects on monocytes remain to be determined. Herein, we investigated the effects of Rh1 on the expression of MCP-1 and CCR2, activation of MAPK signaling, and chemotaxis of monocytes. Treatment of Rh1 decreased the levels of MCP-1 and CCR2 and the expression of VLA5 and activated ß1 integrin on the cell surface, and attenuated the phosphorylation of MAPKs. Based on these results, the inhibitory effects of Rh1 on monocyte function should be regarded as a promising new anti-inflammatory response with a potential therapeutic role against inflammation-dependent diseases.

(-)-Epigallocatechin-3-gallate inhibits invasion and migration of salivary gland adenocarcinoma cells.

(-)-Epigallocatechin-3-gallate (EGCG) has inhibitory effect on a variety of cancers by inducing apoptosis and cell cycle arrest or inhibiting angiogenesis and metastasis. EGCG has been found to induce apoptosis in salivary gland carcinoma cells, however, it is not known whether EGCG affects invasion and migration. Thus, this study was performed to clarify whether EGCG affects invasion and migration of salivary gland tumors. Matrigel invasion assay, wound scratch assay and migration assay using commercial kit were performed. beta1 integrin expression and activation of its downstream molecules such as focal adhesion kinase (FAK), AKT and extracellular signal-regulated kinase (ERK) were examined by Western blot. Enzymatic activity of matrix metalloprotease (MMP)-2 and MMP-9 was examined by gelatin zymography. EGCG inhibited effectively invasion and migration of SGT cells in a dose-dependent manner. EGCG also inhibited the activation of beta1 integrin-downstream molecules such as FAK, AKT and ERK as well as the expression of beta1 integrin itself. Moreover, MMP-2 and MMP-9 expression and their enzymatic activity were reduced by EGCG in a dose-dependent manner. These results indicate that EGCG may effectively suppress salivary gland tumors by inhibiting metastasis through beta1 integrin-mediated signaling.

TM14 is a new member of the fibulin family (fibulin-7) that interacts with extracellular matrix molecules and is active for cell binding.

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin beta1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.

Chitosan monomer accelerates alkaline phosphatase activity on human osteoblastic cells under hypofunctional conditions.

Chitosan is a natural polyaminosaccharide that is extensively applied as an antitumor and antirheumatic drug. However, there are few reports about its effects on hypofunctional osteoblasts in vitro. We investigated the biological characteristics of a human osteoblastic cell line (NOS-1 cells) that was cultured with a chitosan monomer-containing medium under simulated microgravity conditions. After 7 days of cell incubation under the conventional conditions, the flasks were transferred to a microgravity simulator for 3 days. In the 0.005% chitosan monomer supplemented group, the marker enzyme of biological mineralization, the alkaline phosphatase (ALP) activity, was significantly higher compared with the control group (p<0.05). A cDNA microarray was performed to investigate the effects on the mRNA level by chitosan monomer, and the fluorescent signal was analyzed. The interferon gamma (IFN-gamma) receptor gene was detected with a signal ration of 2.2. The slight increase of IFN-gamma receptor expression was confirmed after 3 days of incubation according to RT-PCR analysis. Western blot analysis also showed the increased expression of IFN-gamma receptor. These results suggest that a supra-low concentration of chitosan monomer may increase the ALP activity of osteoblastic cells through the IFN-gamma receptor at the early phase of cell culture and recover the activity for biological mineralization under the hypofunctional condition.